The present invention relates to metabolic disorders or diseases, their prediction, diagnosis, prevention, and treatment. The invention in particular relates to IRS-2 related metabolic disorders or diseases, such diabetes and obesitas, and makes available specific nucleotide sequences and their therapeutic and diagnostic use, as well as their use as research tools, inter alia a novel method and assay for screening drug candidates and for differentiating between and evaluating insulin regulating substances.
Metabolic disorders or diseases are conditions, where the distribution of nutrients and their use, including the elimination of wastes, in a living body is disturbed or disrupted. IRS-2 related metabolic disorders or diseases are abnormal conditions, where the activation, expression or other function of the IRS-2 gene is involved.
Diabetes mellitus is a complex disorder of carbohydrate, fat, and protein metabolism that is primarily a result of a relative or complete lack of insulin secretion by the beta cells of the pancreas or a result of defects of the insulin receptors. The various forms of diabetes are divided in two categories, the most frequent being juvenile-onset diabetes or Type I insulin-dependent diabetes mellitus (IDDM) and adult-onset diabetes or Type II non-insulin-dependent diabetes mellitus (NIDDM), Both diseases, even when correctly diagnosed and medicated, require life-long medication, good patient compliance, a careful diet and frequent medical observation to avoid potentially serious sequelae.
Thiazolidinediones (TZD) are a recently identified class of antidiabetic agents which act by improving insulin sensitivity in both different animal models of obesity and diabetes as well as in man. In addition to improving the glucose and insulin levels, the circulating free fatty acids (FFA) and triglycerides are also lowered.
TZD promote fat cell differentiation and activate several adipocyte-specific genes such as the fatty acid binding protein, aP2, as well as the lipoprotein lipase. There is much recent evidence that TZD induce their diverse effects by binding to and activating the peroxisome proliferator activated receptor (PPAR)xcex3 (reviewed in Spiegelman, 1998). PPARxcex3 is mainly expressed in the adipose tissue and exists as two isoforms, PPARxcex31 and xcex32. PPARxcex31 is the major isoform and accounts for around 85% of that in the adipose tissue. The isoforms differ in their NH2-terminal end, with PPARxcex32 having additional 30 amino acids, and are generated from the same gene by mRNA splicing.
It is currently unclear how TZD improve insulin sensitivity since known PPARxcex3-regulated genes mainly involve adipocyte differentiation, lipid storage and metabolism. It is also noted, however not yet explained, that a number of patients fail to respond to TZD treatment. The percentage vanes between different demographic groups, but may on average amount to as much as 30% of all patients. Due to the complex nature of the disease, the fact that a certain patient fails to respond to the treatment will not become evident until after several weeks of treatment and re-admission or a repeated visit to the physician.
Current hypotheses of the mechanisms for the direct insulin regulating or sensitizing effect of TZD include; the formation of new, small and insulin-sensitive fat cells; the inhibition of TNFxcex1 production and, hence, its negative effects on insulin signaling or, as found in some experiments, increased GLUT4 expression in adipocytes although this requires activation of C/EBPxcex1, as well. However, no clear and reproducible link to the intracellular signaling molecules for insulin has been found so far.
In U.S. Pat. No. 5,478,852, Olefsky et al. disclose methods of using thiazolinedione derivatives and related antihypoglycemic agents in the treatment of impaired glucose tolerance in order to prevent or delay the onset of noninsulin-dependent diabetes mellitus. This reference is however silent on the underlying mechanism of action, responsible for the treatment effects disclosed therein.
In WO 98/21592, Smith and Rondinone disclose methods for screening candidate bioactive agents capable of modulating the activity of IRS-1 and IRS-2, collectively called IRS-molecules. The methods are characterised in that a candidate bioactive agent is added to a sample of the IRS-molecule, whereafter the binding of said candidate agent to said IRS-molecule is determined. Alternatively, the activity of the IRS-molecules is measured before and after the addition of the candidate agent. Neither coding nor non-coding sequences related to IRS-2 are disclosed.
In U.S. Pat. No. 5,858,701, White et al. disclose a nucleic acid sequence encoding an IRS-2 polypeptide, claiming to have discovered the existence of a family of IRS-2 like genes, which share functional and structural properties. These coding sequences, which notably may exhibit variations between population groups and even individuals, are suggested for use in gene therapy, diagnosis of disease, determination of the risk of developing a disease, evaluation of an effect of a treatment, and evaluation of a compound for the ability to bind a nucleic acid encoding an IRS regulatory sequence.
Obviously, the IRS-protein itself and its corresponding coding sequence has been studied and used as the marker, either directly by measuring its activity or concentration, or indirectly, by studying its interaction with other molecules. The non-coding sequences have however, to the best knowledge of the present inventor, hitherto not been identified as such neither suggested for use for research, screening, diagnostic nor therapeutic purposes.
There remains a need to improve the treatment of metabolic, IRS-2 related disorders, such as diabetes and in particular to develop new pharmaceuticals for this purpose, e.g. improved methods for differentiating between insulin regulating, i.e. insulin sensitizing or inhibiting substances. In particular, new, specific, and reliable markers for IRS-2 transcription are needed. One problem to be solved is therefor how to efficiently, and accurately screen candidate drugs, e.g. the class of TZD compounds in respect of insulin regulating, i.e. insulin sensitizing or inhibiting properties in different cells. Another problem is to find genetic markers with wide applicability, useful for the diagnosis of diseases related to abnormal IRS-2 transcription and for the determination of the risk of developing such diseases.
Further problems and their solutions will become evident or possible to deduce from the following description, example and claims.
The above problems and shortcomings of the prior art are solved by invention as set forth in the attached sequence listing and claims, which are hereby incorporated in their entirety. The present inventor has identified specific non-coding sequences upstream to the known IRS-2 gene. These are disclosed in the attached sequence listing (PatentIn 2.1) and numbered SEQ.ID.NO. 1-12. By using at least one of these sequences or closely homologous sequences as the reporter(s) for the evaluation of IRS-2 activation, a more practical and generally applicable assay system is made possible. Similarly, by using at least one of these sequences or closely homologous sequences as the reporter(s) for the evaluation of IRS-2 activation, a more accurate prediction and diagnosis is made possible. Further advantages, embodiments and characteristics of the invention will be evident from the following description and examples.